A comparative study of the patterns of LH and FSH released and synthesized during the normal estrous cycle of the rat was performed in vitro. Groups of female rats were killed at 09.00 h and 15.00 h throughout the 4-day estrous cycle and the adenohypophysis incubated for 4 h. Pituitary extracts and media were assayed for LH and FSH by radioimmunoassay. Both hormones exhibited maximum concentrations in tissue and medium on proestrus afternoon. The increased release of LH occurred during the day of proestrus, while that of FSH lasted from the afternoon of proestrus to the morning of estrus. Synthesis of LH exhibited a marked rising phase from the afternoon of diestrus-2 through the morning of estrus. When release and synthesis of LH and FSH were expressed as percentages of hormone concentration at the beginning of the incubation period, the percentages of FSH were greater than those of LH at all stages of the cycle. Addition of synthetic gonadotrophin-releasing hormone (GnRH) to the incubation medium was followed by a dose-dependent increase in release and synthesis of both LH and FSH. Pituitary responsiveness to GnRH reached a peak on the afternoon of proestrus and fell to a minimum during diestrus. Whereas maximum LH responsiveness correlated with the proestrus discharge of the hormone, that of FSH ceased before the basal release of the hormone, which terminates on estrus. The possible role of sex steroid hormones in regulating in vitro secretion of gonadotrophins and pituitary sensitivity to exogenous GnRH is discussed.